Methods and compositions for the production of monoclonal antibodies

ABSTRACT

The present invention comprises compositions and methods for making monoclonal antibodies. The present invention further comprises vectors that replicate the immune system components, particularly an antigen-presenting cell (APC) element of the immune synapse. Additionally, the present invention may further comprise synthetic T-cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/004,623 filed Dec. 2, 2004 now abandoned which claims priority toU.S. Provisional Application No. 60/526,360 filed Dec. 2, 2003.

FIELD OF THE INVENTION

The present invention relates generally to immunology. The presentinvention further relates to methods and compositions for the productionof monoclonal antibodies and in vitro methods for production of suchantibodies.

BACKGROUND OF THE INVENTION

The introduction of desired agents into specific target cells has been achallenge to scientists for a long time. The challenge of specifictargeting of agents is to get an adequate amount of the agent or thecorrect agent to the target cells of an organism without providing toomuch exposure of the rest of the organism. A very desired target fordelivery of specific agents is the immune system. The immune system is acomplex response system of the body that involves many different kindsof cells that have differing activities. Activation of one portion ofthe immune system usually causes a variety of responses due to unwantedactivation of other related portions of the system. Currently, there areno satisfactory methods or compositions for producing a specificallydesired response by targeting the specific components of the immunesystem.

The immune system is a complex interactive system of the body thatinvolves a wide variety of components, including cells, and cellularfactors, which interact with stimuli from both inside the body andoutside the body. Aside from its direct action, the immune system'sresponse is also influenced by other systems of the body including thenervous, respiratory, circulatory, and digestive systems.

One of the better-known aspects of the immune system is its ability torespond to foreign antigens presented by invading organisms, cellularchanges within the body, or from vaccination. Some of the first kinds ofcells that respond to such activation of the immune system arephagocytes and natural killer cells. Phagocytes include among othercells, monocytes, macrophages, and polymorphonuclear neutrophils. Thesecells generally bind to the foreign antigen, internalize it and oftentimes destroy it. They also produce soluble molecules that mediate otherimmune responses, such as inflammatory responses. Natural killer cellscan recognize and destroy certain virally-infected embryonic and tumorcells. Other factors of the immune response include complement pathways,which are capable of responding independently to foreign antigens oracting in concert with cells or antibodies.

One of the aspects of the immune system that is important forvaccination is the specific response of the immune system to aparticular pathogen or foreign antigen. Part of the response includesthe establishment of “memory” for that foreign antigen. Upon a secondaryexposure, the memory function allows for a quicker and generally greaterresponse to the foreign antigen. Lymphocytes in concert with other cellsand factors play a major role in both the memory function and theresponse.

Generally, it is thought that the response to antigens involves bothhumoral responses and cellular responses. Humoral immune responses aremediated by non-cellular factors that are released by cells and whichmay or may not be found free in the plasma or intracellular fluids. Amajor component of a humoral response of the immune system is mediatedby antibodies produced by B lymphocytes. Cell-mediated immune responsesresult from the interactions of cells, including antigen presentingcells and B lymphocytes (B cells) and T lymphocytes (T cells).

One of the most widely employed aspects of the immune responsecapabilities is the production of monoclonal antibodies. The advent ofmonoclonal antibody (Mab) technology in the mid 1970s provided avaluable new therapeutic and diagnostic tool. For the first time,researchers and clinicians had access to unlimited quantities of uniformantibodies capable of binding to a predetermined antigenic site andhaving various immunological effector functions. Currently, thetechniques for production of monoclonal antibodies are well known in theart.

These monoclonal antibodies are thought to hold great promise inmedicine and diagnostics. Unfortunately, the development of therapeuticproducts based on these proteins has been limited because of problemsthat are inherent in monoclonal antibody therapy. For example, mostmonoclonal antibodies are mouse derived and, thus, do not fix humancomplement well. They also lack other important immunoglobulinfunctional characteristics when used in humans.

The biggest drawback to the use of monoclonal antibodies is the factthat nonhuman monoclonal antibodies are immunogenic when injected into ahuman patient. After injection of a foreign antibody, the immuneresponse mounted by a patient can be quite strong. The immune responsecauses the quick elimination of the foreign antibody, essentiallyeliminating the antibody's therapeutic utility after an initialtreatment. Unfortunately, once the immune system is primed to respond toforeign antibodies, later treatments with the same or different nonhumanantibodies can be ineffective or even dangerous.

Mice can be readily immunized with foreign antigens to produce a broadspectrum of high affinity antibodies. However, the introduction ofmurine antibodies into humans results in the production of ahuman-anti-mouse antibody (HAMA) response due to the presentation of amouse antibody in the human body. Use of murine antibodies in a patientis generally limited to a term of days or weeks. Longer treatmentperiods may result in anaphylaxis. Moreover, once HAMA has developed ina patient, it often prevents the future use of murine antibodies fordiagnostic or therapeutic purposes.

To overcome the problem of HAMA response, researchers have attemptedseveral approaches to modify nonhuman antibodies, to make themhuman-like. These approaches include mouse/human chimers, humanization,and primatization. Early work in making more human-like antibodies usedcombined rabbit and human antibodies. The protein subunits ofantibodies, rabbit Fab fragments and human Fc fragments, were joinedthrough protein disulfide bonds to form new, artificial proteinmolecules or chimeric antibodies.

Recombinant molecular biological techniques have been used to createchimeric antibodies. Recombinant DNA technology was used to construct agene fusion between DNA sequences encoding mouse antibody variable lightand heavy chain domains and human antibody light chain (LC) and heavychain (HC) constant domains to permit expression of chimeric antibodies.These chimeric antibodies contain a large number of nonhuman amino acidsequences and are immunogenic to humans. Patients exposed to thesechimeric antibodies produce human-anti-chimera antibodies (HACA). HACAis directed against the murine V region and can also be directed againstthe novel V-region/C-region (constant region) junctions present inrecombinant chimeric antibodies.

To overcome some of the limitations presented by the immunogenicity ofchimeric antibodies, molecular biology techniques are used to createdhumanized or reshaped antibodies. The DNA sequences encoding the antigenbinding portions or complementarity determining regions (CDRs) of murinemonoclonal antibodies are grafted, by molecular means, on the DNAsequences encoding the frameworks of human antibody heavy and lightchains. The humanized Mabs contain a larger percentage of human antibodysequences than do chimeric Mabs. The end product, which comprisesapproximately 90% human antibody and 10% mouse antibody, contains amouse binding-site on a human antibody. It also contains certain aminoacid substitutions from the mouse Mab into the framework of thehumanized Mab in order to retain the correct shape, and thus, bindingaffinity for the target antigen.

In practice, simply substituting murine CDRs for human CDRs is notsufficient to generate efficacious humanized antibodies retaining thespecificity of the original murine antibody. There is an additionalrequirement for the inclusion of a small number of critical murineantibody residues in the human variable region. The identity of theseresidues depends upon the structure of both the original murine antibodyand the acceptor human antibody. It is the presence of these murineantibody residues that helps create a HACA response in the patient,leading to rapid clearance of the monoclonal antibodies and the fear ofanaphylaxis.

Another technique, called resurfacing technology, is used for humanizingmouse antibodies. Resurfacing involves replacing the mouse antibodysurface with a human antibody surface in a process that is faster andmore efficient than other humanization techniques. This techniqueprovides a method of redesigning murine monoclonal antibodies toresemble human antibodies by humanizing only those amino acids that areaccessible at the surface of the V-regions of the recombinant F_(v). Theresurfacing of murine monoclonal antibodies may maintain the avidity ofthe original mouse monoclonal antibody in the reshaped version, becausethe natural framework-CDR interactions are retained. Again, theseantibodies suffer from the problem of being antigenic due to their mouseorigins.

Other technologies use primate, rather than mouse, sequences to humanizeMabs. The rationale of this approach, called primatization, is that mostof the sequences in the primate antibody variable region areindistinguishable from human sequences. Primatized anti-CD4 Mabs for thetreatment of rheumatoid arthritis and severe asthma are being developed.However, these Mabs are still foreign proteins to the immune system ofthe patient and evoke an immune response.

In an effort to avoid the immune response to foreign proteins, a varietyof approaches are being developed to make human Mabs that contain onlyhuman antibody components. One approach is to isolate a human B cellclone that naturally makes antibody to the desired antigen and to growit in a trioma cell culture system. Because human antibodies are madeonly against antigens that are foreign to the host, none of the human Bcells will make antibodies against human antigens. Therefore, thisapproach is not useful to produce Mabs against antigens that are humanproteins.

Two other approaches to create human Mabs are phage display and use oftransgenic mice. Phage display technique takes advantage of the abilityof humans to make antibodies against any possible structure. Thistechnique uses the antibody genes from many individual humans to createa large library of phage antibodies, each displaying a functionalantibody variable domain on its surface. From this library, individualvariable domains are selected for their ability to bind to the desiredantigen. The Mab is created through molecular biology techniques bycombining an antibody variable domain having the desired bindingcharacteristics and a constant domain that best meets the potentialhuman therapeutic product. Again, this technique lacks antigenspecificity. The phage library cannot contain every binding region forany and all desired antigens. It also may contain binding regions, whichlack specificity. Thus, this technique may require considerableengineering to increase antibody affinities to useful levels.

Transgenic mice are also being used to create “human” antibodies. Thetransgenic mice are created by replacing mouse immunoglobulin gene lociwith human immunoglobulin loci. This approach may provide advantagesover phage display technologies because it takes advantages of mouse invivo affinity maturation machinery.

All of the current technologies for producing human or human-like Mabsare insufficient to provide a species-specific antibody that is antigenspecific for a described antigen. Chimeric antibodies have theadvantages of retaining the specificity of the murine antibody andstimulating human Fc dependent complement fixation and cell-mediatedcytotoxicity. However, the murine variable regions of these chimericantibodies can still elicit a HAMA response, thereby limiting the valueof chimeric antibodies as diagnostic and therapeutic agents.

Vaccines may be directed at any foreign antigen, whether from anotherorganism, a changed cell, or induced foreign attributes in a normal“self” cell. The route of administration of the foreign antigen can helpdetermine the type of immune response generated. For example, deliveryof antigens to mucosal surfaces, such as oral inoculation with livepolio virus, stimulates the immune system to produce an immune responseat the mucosal surface. Injection of antigen into muscle tissue oftenpromotes the production of a long lasting IgG response.

Vaccines may be generally divided into two types, whole and subunitvaccines. Whole vaccines may be produced from viruses or microorganismswhich have been inactivated or attenuated or have been killed. Liveattenuated vaccines have the advantage of mimicking the naturalinfection enough to trigger an immune response similar to the responseto the wild-type organism. Such vaccines generally provide a high levelof protection, especially if administered by a natural route, and somemay only require one dose to confer immunity. Another advantage of someattenuated vaccines is that they provide person-to-person passage amongmembers of the population. These advantages, however, are balanced withseveral disadvantages. Some attenuated vaccines have a limitedshelf-life and cannot withstand storage in tropical environments. Thereis also a possibility that the vaccine will revert to the virulentwild-type of the organism, causing harmful, even life-threatening,illness. The use of attenuated vaccines is contraindicated inimmunodeficient states, such as AIDS, and in pregnancy.

Killed vaccines are safer in that they cannot revert to virulence. Theyare generally more stable during transport and storage and areacceptable for use in immunocompromised patients. However, they are lesseffective than the live attenuated vaccines, usually requiring more thanone dose. Additionally, they do not provide for person-to-person passageamong members of the population.

Production of subunit vaccines requires knowledge about the epitopes ofthe microorganism or cells to which the vaccine should be directed.Other considerations in designing subunit vaccines are the size of thesubunit and how well the subunit represents all of the strains of themicroorganism or cell. The current focus for development of bacterialvaccines has shifted to the generation of subunit vaccines because ofthe problems encountered in producing whole bacterial vaccines and theside effects associated with their use. Such vaccines include a typhoidvaccine based upon the Vi capsular polysaccharide and the Hib vaccine toHaemophilus influenzae.

Because of the safety concerns associated with the use of attenuatedvaccines and the low efficacy of killed vaccines, there is a need in theart for compositions and methods that enhance vaccine efficacy. There isalso a need in the art for compositions and methods of enhancing theimmune system, which stimulate both humoral and cell-mediated responses.There is a further need in the art for the selective adjustment of animmune response and manipulating the various components of the immunesystem to produce a desired response. Additionally, there is a need formethods and compositions that can accelerate and expand the immuneresponse for a more rapid activation response. There is an increasedneed for the ability to vaccinate populations, of both humans andanimals, with vaccines that provide protection with just one dose.

What is needed are compositions and methods to target the delivery ofspecific agents to only the target cells. Such compositions and methodsshould be able to deliver therapeutic agents to the target cellsefficiently. What is also needed are compositions and methods that canbe used both in in vitro and in vivo systems.

There is also a general need for compositions of monoclonal antibodiesand improved methods for producing them. There is a particular need formethods for producing human antibodies having affinity for apredetermined antigen. These human immunoglobulins should be easily andeconomically produced in a manner suitable for therapeutic anddiagnostic formulation.

SUMMARY OF THE INVENTION

The present invention comprises compositions and methods for makingspecies-specific antigen-specific monoclonal antibodies, preferably IgGmonoclonal antibodies. The present invention further comprises vectorsthat replicate elements of the immune system, particularly theantigen-presenting cell (APC) element of the immune synapse. A preferredvector optionally comprises binding an antigen-loaded majorhistocompatibility (MHC) class II protein, the co-stimulatory proteinB7, and the structural protein intracellular adhesion protein (I-CAM)onto the surface of colloidal metal vectors. Such vectors replicate the3-D orientation of the APC (FIG. 3) generating a syntheticantigen-presenting cell (sAPC) capable of activating CD4⁺ T-cells tomature the antibody response of immunized B-cells.

The present invention further comprises vectors, including a syntheticCD4⁺ T-cell (sTc), and a synthetic germinal center (sGC). In oneembodiment the synthetic CD4⁺ T-cell is comprised of colloidal metalvectors bound with CD40 ligand and cytokines. In another embodiment thesynthetic germinal center is comprised of colloidal metal vectors boundwith B Lymphocyte Stimulator; BlyS and CD30L/receptor system, thatincrease the efficiency and specificity of B-cell antibody response toin vitro immunization. While not wishing to be bound to any particulartheory, in one embodiment the physical juxtaposition of the antigen withB-cell growth factors improves the uptake of the human TNF antigenthrough the surface IgM antigen receptor and induces a more robustB-cell response. Having these signals juxtaposed on the same B-cellfurther improves the ability to elicit an antigen specific B-cellresponse in vitro.

The present invention comprises methods of making the synthetic immunecomponent elements. Methods are taught herein for making vectorcompositions that mimic the functionality of components of the immunesystem. The present invention also comprises methods of treatment ofimmune system-related diseases and pathologies. Methods of vaccinationare also included in the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic representation of the immune synapse.

FIG. 2 provides a schematic representation of the differentiation ofprimary antibody response by activated CD4⁺T-cell.

FIG. 3A provides a schematic of the colloidal gold syntheticantigen-presenting cell. FIG. 3B provides a schematic of the colloidalgold synthetic T-Cell. FIG. 3C provides a schematic of the colloidalgold synthetic germinal center.

FIG. 4 provides a schematic representation of the inability of a singleparticle sAPC to form a functional immune synapse.

FIG. 5 provides a schematic representation of the generation of amultiple particle colloidal gold sAPC.

FIG. 6 provides a graph depicting the binding multiple cytokines to thesame particle of colloidal gold.

FIG. 7 is a series of photographs of EGF streptavidin gold that wastargeted to macrophages (FIG. 7A), dendritic cells (FIG. 7B) and B-Cells(FIG. 7C).

FIG. 8 provides a graph of the immunoreactivity of cells in response tovarious stimuli in vitro.

FIG. 9A provides a schematic of the self-assembly of colloidal goldparticle on the solid support of an EIA plate. 1=EIA plate; 2=Murine Mabagainst human TNF; 3=human TNF (blue box); 4=32 nm colloidal gold boundwith streptavidin an TNF; 5=biotinylated BSA; 6=17 nm streptavidincolloidal gold; 7=biotinylated human IL-6; 8=alkaline phosphataseconjugated rabbit anti-human IL-6.

FIG. 9B provides a schematic of self-assembly of colloidal goldparticles bound with either IL-1 or TNF on a four-arm PEG-thiol backbone(Sun Bio, Inc.).

FIG. 10A provides a graph of the immunoreactivity signal generated bythe particle in FIG. 9A.

FIG. 10B provides a graph of the immunoreactivity signal generated bythe particle in FIG. 9B.

FIG. 11 provides a schematic representation of the colloidal gold/TNFbinding apparatus

FIG. 12 provides a graph of the effect of ionic strength on thestability of the colloidal gold TNF vector after lyophilization.

FIG. 13 provides a schematic representation of a model for TNF bindingto colloidal gold in low ionic strength solutions.

FIG. 14 provides a schematic representation of a model for TNF bindingto colloidal gold at high ionic strength solutions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of specific embodiments included herein.Although the present invention has been described with reference tospecific details of certain embodiments, thereof, it is not intendedthat such details should be regarded as limitations upon the scope ofthe invention. The entire text of the references mentioned herein arehereby incorporated in their entireties by reference, including U.S.Provisional Application Ser. No. 60/526,360.

The present invention comprises methods and compositions for generatingantigen specific, species-specific IgG monoclonal antibodies. Thepresent invention comprises methods and compositions comprisingnaturally occurring and/or synthetic vectors that replicate theantigen-presenting cell (APC), T cell and germinal center elements ofthe humoral immune response.

The present invention comprises vectors that mimic any of the elementsor stages of the immune response. The immune response is initiated bythe recognition of foreign antigens by various kinds of cells,principally macrophages or other antigen presenting cells. This leads toactivation of lymphocytes, in particular, the lymphocytes thatspecifically recognize that particular foreign antigen and results inthe development of the immune response, and possibly, elimination of theforeign antigen. Overlaying the immune response directed at eliminationof the foreign antigen are complex interactions that lead to helperfunctions, stimulator functions, suppresser functions and otherresponses. The power of the immune system's responses must be carefullycontrolled at multiple sites for stimulation and suppression or theresponse will either not occur, over respond, or not cease afterelimination.

The recognition phase of response to foreign antigens consists of thebinding of foreign antigens to specific receptors on immune cells. Thesereceptors generally exist prior to antigen exposure. Recognition canalso include interaction with the antigen by macrophage-like cells or byrecognition by factors within serum or bodily fluids.

In the activation phase, lymphocytes undergo at least two major changes.They proliferate, leading to expansion of the clones of antigen-specificlymphocytes and amplification of the response, and the progeny ofantigen-stimulated lymphocytes differentiate either into effector cellsor into memory cells that survive, ready to respond to re-exposure tothe antigen. There are numerous amplification mechanisms that enhancethis response.

In the effector phase, activated lymphocytes perform the functions thatmay lead to elimination of the antigen or establishment of the vaccineresponse. Such functions include cellular responses, such as regulatory,helper, stimulator, suppressor or memory functions. Many effectorfunctions require the combined participation of cells and cellularfactors. For instance, antibodies bind to foreign antigens and enhancetheir phagocytosis by blood neutrophils and mononuclear phagocytes.Complement pathways are activated and may participate in the lysis andphagocytosis of microbes in addition to triggering other body responses,such as fever.

In the immune response to antigens, immune cells interact with eachother by direct cell-to-cell contact or indirect cell-to-cell (factormediated) communication. For example, interactions between T cells,macrophages, dendritic cells, and B cells are necessary for an effectiveimmune response. Antigen-presenting cells (APC) activate B and T cellsby presenting them B and T cells with processed antigens and otheractivation signals. Activated T cells help control immune responses andparticipate in the removal of foreign organisms. Helper T cells causecells to become better effector cells, such as helping cytotoxic T cellprecursors to develop into killer cells, helping B cells makeantibodies, and helping increase functions of other cells likemacrophages. Activated B cells divide and produce antigen specificantibodies and memory B cells. The cells involved in the immune responsealso secrete cellular factors or cytokines, which enhance the functionsof phagocytes, stimulate inflammatory responses and affect a variety ofcells.

The reactions of these cells also involve feedback loops. Macrophagesand other mononuclear phagocytes, or APCs, actively phagocytose antigensfor presentation to B and T cells and such activity can be enhanced bylymphocytic cellular factors. Macrophages also produce cytokines that,among other activities, stimulate T cell proliferation anddifferentiation, recruit other inflammatory cells, especiallyneutrophils, and are responsible for many of the systemic effects ofinflammation, such as fever. One such cytokine, called interleukin-12,is especially important for the development of cell-mediated immunity.

Dendritic cells are also APCs, which initiate an immune response. Thereare a number of different types of dendritic cells, including lymphoiddendritic cells and Langerhans cells of the skin. They can be foundthroughout the body and particularly in the spleen, lymph nodes,tonsils, Peyer's patches, and thymus. They are irregularly shaped cells,which continuously extend and contract dendritic (tree-like) processes.One of their roles in the immune system is to induce and regulate B andT cell activation and differentiation. They are potent accessory cellsfor the development of cytotoxic T cells, antibody formation by B cells,and some polyclonal responses like oxidative mitogenesis. They alsostimulate T cells to release the cytokine interleukin-2

An important arm of vaccination is the response to antigens that isprovided by B lymphocytes or B cells. B cells represent about 5 to 15%of the circulating lymphocytes. B cells produce immunoglobulins, IgG,IgM, IgA, IgD, and IgE, which may be released into body fluids, secretedwith attached proteins or be inserted into the surface membrane of the Bcell. Such immobilized immunoglobulins act as specific antigenreceptors. In responding to antigen, these immunoglobulin receptors arecrosslinked at a specific site on the B cell. This process, which isknown as capping, is followed by internalization and degradation of theimmunoglobulin. In APCs, which may include B cells, antigen fragmentsare combined with the MHC and ultimately expressed on the surface of theAPC.

The B plasma cells produce and secrete antibody molecules that can bindforeign proteins, polysaccharides, lipids, or other chemicals inextracellular or cell-associated forms. The antibodies produced by asingle plasma cell are specific for one antigen. The secreted antibodiesbind the antigen and trigger the mechanisms that facilitate theirdestruction.

In 1975, Kohler and Milstein (Kohler, G., and Milstein, C., Nature(London). 1975. volume 256: pp-495) described a method for fusingantibody-producing B cells isolated from the spleens of immunized micewith aggressively proliferating mouse myeloma cells. This resultanthybrid cell, a hybridoma, possesses the characteristics of both parentalcells. It produces and secretes large amounts of antibody during itscontinued growth and proliferation. Through a series of systematiccellular dilutions, genetically singular hybridoma cells can be isolatedthat produce an antibody of singular specificity, a monoclonal antibody(Mab).

The most common procedures require that the production of monoclonalantibodies start with the immunization of an animal. Antigen, draininginto a local lymph node or spleen, activates naïve B-cells to produceIgM antibodies. These activated B cells are then presented withantigen-activated CD4⁺ T cells to induce class switching. Classswitching is characterized by a change in the production of antibodytype from IgMs to IgGs (Kuby, J., Immunology Third Edition 1997. edsAllen D., pp-205-213). Antibody secreting B cell lymphocytes areisolated from the lymph node or spleen of the immunized animal, and arefused with species-specific myeloma cells. The fused cells are thenallowed to grow to produce antigen specific IgG antibodies. During thescreening process, positive fusion clones are selected for theirtherapeutic potential.

Mice can be readily immunized with foreign antigens to produce a broadspectrum of high affinity antibodies. However, the introduction ofmurine antibodies into humans results in the production of ahuman-anti-mouse antibody (HAMA) response due to the presentation of aforeign protein in the body. Use of murine antibodies in a patient isgenerally limited to a term of days or weeks. Moreover, once HAMA hasdeveloped in a patient, it often prevents the future use of murineantibodies for other diagnostic or therapeutic purposes.

The early success of this technology in animals prompted scientists inthe 1980's to extend this concept and attempt to produce humanmonoclonal antibodies. However, extrapolation from animal to human wasfraught with difficulties. The first hurdle was the lack of antigenspecific B cells. Standard monoclonal antibody procedure requires thatthese cells be harvested from an animal that had been immunized, amethod not generally applicable to humans. This problem is furthercompounded by (i) the fact that there is no ready source of activated Bcells, (ii) the paucity of immune competent B cells present inperipheral blood, and (iii) the inability to obtain either lymph nodesor spleens from human subjects. These factors prompted the developmentof a variety in vitro strategies to produce human monoclonal antibodies.Although initial results showed great promise, the inability tocompletely reconstruct the sequence of events of the in vivo antibodyresponse ultimately caused the technology to fail and this technicalapproach has been essentially abandoned.

The first barrier to in vitro antibody production is the relatively lowconversion rate of naïve human B cell lymphocytes to activated B cells.In the past resolving this challenge proved difficult even when recallantigens, such as Tetanus toxin (Butler et. al., J. Immuol. 1983. volume130: pp-165), were used to induce a primary antibody response from humanperipheral blood B cell lymphocytes. The present invention comprisesmethods for making vectors that activate pathways that lead to antibodygeneration. The present invention also comprises compositions ofnaturally occurring or synthetic vectors. Such vectors comprisecolloidal gold platforms with multiple B cell ligands associated.

Numerous examples of cross-linking of receptor/ligand pairs topotentiate biologic responses have been described (Carroll, K., Prosser,E., and Kennedy, R. Hybridoma 1991. 10: 229-239). The present inventioncomprises vectors of colloidal metal that increase the efficiency andspecificity of B cell antibody response to in vitro immunization. Thoughnot wishing to be bound by any particular theory, it is believed thatthe physical juxtaposition of the antigen with B cell growth factorsimproves the uptake of the antigen through the surface IgM antigenreceptor and induces a more robust B cell response. There is alsoimproved antigen processing and presentation. Having these signalsjuxtaposed on the same B cell improves the ability to elicit an antigenspecific B cell response in vitro.

In one embodiment, the component-specific immunostimulating moleculeand/or MHC protein and/or the antigen may be bound directly to thecolloidal metal platform or may be bound to the colloidal metal platformthrough members of a binding group. Such binding groups may comprisefree sulfhydryl or pyridyl groups present on, or synthetically added tothe immune component. A preferred embodiment of the present inventioncomprises a colloidal metal as a platform that is capable of binding amember of a binding group to which a component-specificimmunostimulating agent, or a MHC protein or an antigen are bound tocreate a synthetic APC. In an alternatively preferred embodiment, thebinding group is streptavidin/biotin and the component-specificimmunostimulating agent is a cytokine Embodiments of the presentinvention may also comprise binding the antigen, or the MHC protein orthe component-specific immunostimulating agent in a less specificmethod, without the use of binding partners, such as by usingpolycations or proteins. As such, the present invention contemplates theuse of interacting molecules such as polycationic elements known tothose skilled in the art including, but not limited to, polylysine,protamine sulfate, histones or asialoglycoproteins.

The members of the binding pair comprise any such binding pairs known tothose skilled in the art, including but not limited to, antibody-antigenpairs, enzyme-substrate pairs; receptor-ligand pairs; andstreptavidin-biotin. Novel binding partners may be specificallydesigned. An essential element of the binding partners is the specificbinding between one of the binding pair with the other member of thebinding pair, such that the binding partners are capable of being joinedspecifically. Another desired element of the binding members is thateach member is capable of binding or being bound to either anintegrating molecule or a targeting molecule.

The compositions of the invention comprise a colloidal metal, anantigen, and a component specific immunostimulating agent.Alternatively, compositions of the invention comprise a colloidal metal,a MHC protein, an antigen, and a component specific immunostimulatingagent. The component specific immunostimulating agent may comprisebiologically active agents that can be used in therapeutic applicationsor the component specific immunostimulating agent may be useful indetection methods. In additional embodiments, one or more componentspecific immunostimulating agents are admixed, associated with or bounddirectly or indirectly to the colloidal metal. Admixing, associating andbinding includes covalent and ionic bonds and other weaker or strongerassociations that allow for long term or short term association of thederivatized-PEG or the derivatized poly-l-lysine, component specificimmunostimulating agents, and other components with each other and withthe colloidal metal particles.

In yet another embodiment, the compositions may also comprise one ormore targeting molecules admixed, associated or bound to the colloidalmetal. The targeting molecule can be bound directly or indirectly to themetal particle. Indirect binding includes binding through molecules suchas polylysines or other integrating molecules or any association with amolecule that binds to both the targeting molecule and either the metalsol or another molecule bound to the metal sol.

Of particular interest are detection agents such as dyes or radioactivematerials that can be used for visualizing or detecting the sequesteredcolloidal metal vectors. Fluorescent, chemiluminescent, heat sensitive,opaque, beads, magnetic and vibrational materials are also contemplatedfor use as detectable agents that are associated or bound to colloidalmetals in the compositions of the present invention.

Generation of a primary antibody response from naïve human B cells invitro represents only the first step in the in vitro reconstruction ofthe human antibody response. The primary antibody response fromimmunized human B cells results in the secretion of IgM antibodies. Asecond class of lymphoid cells, known as antigen presenting cells(APCs), also internalizes the antigen. Once internalized these cellsprocess the protein antigen into fragments, which are then expressed onthe cell's surface bound to one of two major histocompatibilitycomplexes (MHCs). These cells are important for antibody classswitching.

A current theory of immune system responses is herein presented. Thepresent invention is not limited to the mechanisms described herein, butcan function in multiple methods, not limited by any particular theorydescribed herein. Depending on the microenvironment, APCs expressingantigen bound to class II MHC molecules activate one of two subsets ofCD4⁺ T cells. These cells, also known as helper T cells, perform thenecessary accessory functions to facilitate the cellular or the humoral(antibody) immune response. T_(H)1 CD4⁺ cells facilitate the cellularimmune response, while the T_(H)2 subset of CD4⁺ cells interact with IgMsecreting B cells to initiate the process of class switching.

The activation of CD4⁺ T_(H)2 T-cells by the APC occurs with theformation of a bicellular cleft known as the immune synapse (Wulfing C,Sumen C, Sjaastad M D, Wu L C, Dustin M L, Davis M M. Nat Immunol 2002.31: 42-7). The formation of the immune synapse involves interaction andrearrangement of signaling and structural ligands on the APC with theirrespective receptors on the T cell to form a three-dimensional (3-D)bridge that allows contact and signaling between these two cells (FIG.1). Antigen signaling between the APC and the T cell occurs through thebinding of the MHC/antigen complex with the T cell receptor complex,while the structural integrity of the immune synapse is maintained bythe interaction of ICAM (intracellular adhesion molecule), LFA-3, andCD72 on the APC with LFA-1, CD2, and CD5 receptors on T cells,respectively. The successful formation of the immune synapse causes theCD4⁺ T cell to express a B cell stimulatory molecule known as CD40ligand.

The formation of the immune synapse may signal the T cell to becomeactive or inactive (anergic). Which response is initiated is dependenton the strength of the co-stimulatory signals provided by the B7molecule on the APC to the T cell. The B7 molecule may interact witheither B7 receptor molecule on the T cell, CD28 or CTLA4. These B7receptors differ with respect to their density on the surface of the Tcell as well as their affinity for the B7 molecule. CD28 has a loweraffinity for B7 than CTLA4, but is present at a much higher density onthe surface of the T cell. The binding of B7 to the CD28 receptor sendsan activation signal to the T cell, while the binding of B7 by CTLA4induces T cell anergy (Kuby, J., Immunology Third Edition 1997. edsAllen D., pp. 213-218). Thus, presenting excess B7 in the immune synapsewill ensure that the T cells will be activated. The activated CD4⁺/CD40⁺T cell forms a synapse with an IgM secreting B cell. The interaction ofCD40 ligand on the T cell with the CD40 receptor on the B cell causesthe IgM secreting B cell to undergo class switching to produce IgGs(FIG. 2).

The present invention comprises methods of making sAPCs capable ofactivating CD4+T cells, and synthetic CD4⁺ T-cells (sTc) and syntheticgerminal centers (sGC) able to mature the antibody response of immunizedB cells or immortalized B cells. The compositions of the presentinvention comprise colloidal metal vectors capable of activating T cellsand vectors that cause the maturation of immunized or immortalized Bcells. For example, a vector may have an antigen-loaded majorhistocompatibility (MHC) class II protein, the co-stimulatory proteinB7, and a structural protein such as intracellular adhesion molecule(ICAM), LFA-3 and CD72, associated with the surface of colloidal metalvectors. This vector replicates the 3-D orientation of the APC (FIG. 3)and functions as a synthetic antigen-presenting cell (sAPC) capable ofactivating CD4⁺ T cells to mature the antibody response of immunized Bcells. One embodiment of the sAPC comprises all of the components on asingle particle of colloidal metal. Another embodiment of the sAPCcomprises the constituent proteins of the immune synapse bound onseparate particles of colloidal gold that self-assemble in vitro to formthe sAPC.

The methods and compositions of the present invention comprisingsynthetic antigen-presenting cells (sAPC) comprise compositions that arereadily available and can be “pulled out of the refrigerator” and usedto manipulate the human antibody response. Thus the present inventioncomprises methods of treatment of diseases and immune relateddysfunctions and pathologies. The colloidal metal compositions providecontrol over the variables that are responsible for initiating,maintaining and regulating the immune response (either down-regulatingor up-regulating), such as particle size, the amount of protein boundper particle, the flexibility of protein movement on the particle, aswell as the 3-D assembly of the particles, ensures reproducible controlof the sAPC.

The vector compositions of the present invention can be used in in vitroproduction of monoclonal antibodies. Such monoclonal antibodies can beused in methods of treatment for multiple diseases. The vectorcompositions of the present invention can also be used in makingimproved vaccine compositions.

In vaccine therapy, compositions of synthetic immunogens specificallydesigned to stimulate both the cellular and humoral responses of thehuman immune system are used. By creating specific synthetic cellularimmune elements for the presentation of the antigen and stimulation ofspecific cells, a more predictable and efficient vaccine response isenabled.

The present invention comprises combination vaccines and DNA vaccines.An example of a combination vaccine is the Bordetella pertussis toxinand its surface fimbrial hemaglutinin. In DNA vaccination, the patientis administered nucleic acids encoding a protein antigen that is thentranscribed, translated and expressed in some form to produce strong,long-lived humoral and cell-mediated immune responses to the antigen.

The immune response created by vaccines can be non-specifically enhancedby the use of adjuvants. These are a heterogeneous group of compounds orcarrier components, such as liposomes, emulsions or microspheres, withseveral different mechanisms of action. Methods of the present inventioncomprise use of vaccines for protection against disease, and to treatcancer.

Many diseases, in addition to cancer, are mediated by the immune systemand the present invention comprises methods of treatment of suchdiseases by the administration of an effective amount of a compositioncomprising a colloidal metal vector that is capable of stimulating theimmune system and its components. The diseases include, Crohn's disease,psoriasis, inflammatory bowel disease, adult respiratory distresssyndrome, allergies, eczema, rhinitis, urticaria, anaphylaxis,transplant rejection, such as kidney, heart, pancreas, lung, bone, andliver transplants; rheumatic diseases, systemic lupus erthematosus,rheumatoid arthritis, seronegative spondylarthritides, Sjogren'ssyndrome, systemic sclerosis, polymyositis, dermatomyositis, type 1diabetes mellitus, acquired immune deficiency syndrome, hand foot andmouth disease, Hashimoto's thyroiditis, Graves' disease, Addison'sdisease, polyendocrine autoimmune disease, hepatitis, sclerosingcholangitis, primary biliary cirrhosis, pernicious anemia, coeliacdisease, antibody-mediated nephritis, glomerulonephritis, Wegener'sgranulomatosis, microscopic polyarteritis, polyarteritis nodosa,pemphigus, dermatitis herpetiformis, vitiligo, multiple sclerosis,encephalomyelitis, Guillain-Barre syndrome, myasthenia gravis,Lambert-Eaton syndrome, sclera, episclera, uveitis, chronicmucocutaneous candidiasis, Bruton's syndrome, transienthypogammaglobulinemia of infancy, myeloma, X-linked hyper IgM syndrome,Wiskott-Aldrich syndrome, ataxia telangiectasia, autoimmune hemolyticanemia, autoimmune thrombocytopenia, autoimmune neutropenia,Waldenstrom's macroglobulinemia, amyloidosis, chronic lymphocyticleukemia, and non-Hodgkin's lymphoma.

The present methods enhance vaccine effectiveness by targeting specificimmune components for activation. Compositions comprisingcomponent-specific immunostimulating agents in association withcolloidal metal and antigen are used. Examples of diseases for whichvaccines are currently available include, but are not limited to,cholera, diphtheria, Haemophilus, hepatitis A, hepatitis B, influenza,measles, meningitis, mumps, pertussis, small pox, pneumococcalpneumonia, polio, rabies, rubella, tetanus, tuberculosis, typhoid,Varicella-zoster, whooping cough, and yellow fever.

The combination of route of administration and the vectors used todeliver the antigen to the immune system is a powerful tool in designingthe desired immune response. The present invention comprises methods andcompositions comprising various vectors or vectors in association withdelivery agents, such as liposomes, microcapsules, or microspheres thatcan provide long-term release of immune stimulating vector compositions.These delivery systems act like internal depots for holding the vectorand slowly releasing it for immune system activation. For example, aliposome may be filled with a composition comprising a vector comprisingan antigen and component-specific immunostimulating agents associatedwith colloidal metal.

The antigen/component-specific immunostimulating agent/metal complex isslowly released from the liposome and is recognized by the immune systemas foreign and the specific component to which the component-specificimmunostimulating agent is directed activates the immune system. Thecascade of immune response is activated more quickly by the presence ofthe component-specific immunostimulating agent and the immune responseis generated more quickly and more specifically.

Other methods and compositions contemplated in the present inventioninclude using antigen/component-specific immunostimulatingagent/colloidal metal complexes in which the colloidal metal particleshave different sizes. Sequential administration of component-specificimmunostimulating agents may be accomplished in a one-doseadministration by the use of these differently sized colloidal metalparticles. One dose would include four independent component-specificimmunostimulating agents complexed to an antigen and each with adifferently sized colloidal metal particle. Thus, simultaneousadministration would provide sequential activation of the immunecomponents to yield a more effective vaccine and more protection for thepopulation. Other types of such single dose administration withsequential activation could be provided by combinations of differentlysized colloidal metal particles and liposomes or liposomes filled withdifferently sized colloidal metal particles.

Use of such a vaccination system as described above is very important inproviding vaccines that can be administered in one dose. One doseadministration is important in treating animal populations such aslivestock or wild populations of animals. One dose administration isvital in treatment of populations that are resistant to healthcare suchas the poor, homeless, rural residents or persons in developingcountries that have inadequate health care. Many persons, in allcountries, do not have access to preventive types of health care, suchas vaccination. The re-emergence of infectious diseases, such astuberculosis, has increased the demand for vaccines that can be givenonce and still provide long-lasting, effective protection. Thecompositions and methods of the present invention provide such effectiveprotection.

The term “colloidal metal,” as used herein, includes any water-insolublemetal particle or metallic compound as well as colloids of non-metalorigin such as colloidal carbon dispersed in liquid or water (ahydrosol). Examples of colloidal metals, which can be used in thepresent invention include, but are not limited to, metals in groups HA,IB, IIB and IIIB of the periodic table, as well as the transitionmetals, especially those of group VIII. Preferred metals include gold,silver, aluminum, ruthenium, zinc, iron, nickel and calcium. Othersuitable metals may also include the following in all of their variousoxidation states: lithium, sodium, magnesium, potassium, scandium,titanium, vanadium, chromium, manganese, cobalt, copper, gallium,strontium, niobium, molybdenum, palladium, indium, tin, tungsten,rhenium, platinum, and gadolinium. The metals are preferably provided inionic form (preferably derived from an appropriate metal compound), forexample, the Al³⁺, Ru³⁺, Zn²⁺, Fe³⁺, Ni²⁺ and Ca²⁺ ions. A preferredmetal is silver, particularly in a sodium borate buffer, having theconcentration of between approximately 0.1% and 0.001%, and mostpreferably as approximately a 0.01% solution. Another preferred metal isgold, particularly in the form of Au³⁺. An especially preferred form ofcolloidal gold is HAuCl4 (OmniCorp, South Plainfield, N.J.). The colorof such a colloidal silver solution is yellow and the colloidalparticles may range from 1 to 100 nanometers. Such metal ions may bepresent in the complex alone or with other inorganic ions.

Any antigen may be used in the present invention. Examples of antigensuseful in the present invention include, but are not limited to,Interleukin-1 (“IL-1”), Interleukin-2 (“IL-2”), Interleukin-3 (“IL-3”),Interleukin-4 (“IL-4”), Interleukin-5 (“IL-5”), Interleukin-6 (“IL-6”),Interleukin-7 (“IL-7”), Interleukin-8 (“IL-8”), Interleukin-10(“IL-10”), Interleukin-11 (“IL-11”), Interleukin-12 (“IL-12”),Interleukin-13 (“IL-13”), lipid A, phospholipase A2, endotoxins,staphylococcal enterotoxin B, Pertussis toxin, Tetanus toxin and othertoxins, Type I Interferon, Type II Interferon, Tumor Necrosis Factor(TNF-α or b), Transforming Growth Factor-β (“TGF-β”), Lymphotoxin,Migration Inhibition Factor, Granulocyte-Macrophage Colony-StimulatingFactor (“CSF”), Monocyte-Macrophage CSF, Granulocyte CSF, vascularepithelial growth factor (“VEGF”), Angiogenin, transforming growthfactor (“TGF-α”), heat shock proteins, Epidermal growth factor (“EGF”),carbohydrate moieties of blood groups, Rh factors, fibroblast growthfactor, and other inflammatory and immune regulatory proteins,nucleotides, DNA, RNA, mRNA, sense, antisense, cancer cell specificantigens; such as MART, MAGE, BAGE, and heat shock proteins (HSPs);mutant p53; tyrosinase; mucines, such as Muc-1, PSA, TSH, autoimmuneantigens; immunotherapy drugs, such as AZT; and angiogenic andanti-angiogenic drugs, such as angiostatin, endostatin, basic fibroblastgrowth factor, and vascular endothelial growth factor, prostate specificantigen and thyroid stimulating hormone.

The component-specific immunostimulating agent may be any molecule orcompound which effects the immune system, for example, any molecule thatincreases the APC's ability to stimulate the B cell's production ofantibodies. Examples of component-specific immunostimulating agentsinclude, but are not limited to, antigens, colloidal metals, adjuvants,receptor molecules, nucleic acids, immunogenic proteins, and accessorycytokine/immuostimulators, pharmaceuticals, chemotherapy agents, andcarriers.

Any type of pharmaceutical agent can be employed in the presentinvention. For example, anti-inflammatory agents such as steroids andnonsteroidal anti-inflammatory agents, soluble receptors, antibiotics,analgesic, COX-2 inhibitors. Chemotherapeutic agents of particularinterest include the following non-limiting examples, taxol, paclitaxel,taxanes, vinblastin, vincristine, doxorubicin, acyclovir, cisplatin,methotrexate, mithramycin and tacrine.

These component-specific immunostimulating agents may be employedseparately, or in combinations. They may be employed in a free state orin complexes, such as in combination with a colloidal metal.

Examples of component-specific immunostimulating agents useful in thepresent invention include, but are not limited to, Interleukin-1(“IL-1”), Interleukin-2 (“IL-2”), Interleukin-3 (“IL-3”), Interleukin-4(“IL-4”), Interleukin-5 (“IL-5”), Interleukin-6 (“IL-6”), Interleukin-7(“IL-7”), Interleukin-8 (“IL-8”), Interleukin-10 (“IL-10”),Interleukin-11 (“IL-11”), Interleukin-12 (“IL-12”), Interleukin-13(“IL-13”), lipid A, phospholipase A2, endotoxins, staphylococcalenterotoxin B and other toxins, Type I Interferon, Type II Interferon,Tumor Necrosis Factor (“TNF-α”), Flt-3 ligand, Transforming GrowthFactor-β (“TGF-β”) Lymphotoxin, Migration Inhibition Factor,Granulocyte-Macrophage Colony-Stimulating Factor (“CSF”),Monocyte-Macrophage CSF, Granulocyte CSF, vascular epithelial growthfactor (“VEGF”), Angiogenin, transforming growth factor (“TGF-α”), heatshock proteins, carbohydrate moieties of blood groups, Rh factors,fibroblast growth factor, and other inflammatory and immune regulatoryproteins, nucleotides, DNA, RNA, mRNA, sense, antisense, cancer cellspecific antigens; such as MART, MAGE, BAGE, and heat shock proteins(HSPs); mutant p53; tyrosinase; autoimmune antigens; immunotherapydrugs, such as AZT; and angiogenic and anti-angiogenic drugs, such asangiostatin, endostatin, basic fibroblast growth factor, vascularendothelial growth factor (VEGF) and prostate specific antigen andthyroid stimulating hormone.

Adjuvants useful in the invention include, but are not limited to, heatkilled M. Butyricum and M. Tuberculosis. Nonlimiting examples ofnucleotides are DNA, RNA, mRNA, sense, and antisense. Examples ofimmunogenic proteins include, but are not limited to, KLH (KeyholeLimpet Cyanin), thyroglobulin, and fusion proteins, which have adjuvantand antigen moieties encoded in the gene.

Component-specific immunostimulating agents may be delivered in theirnucleic acid form, using known gene therapy methods, and produce theireffect after translation. Additional elements for activation of immunecomponents, such as antigens, could be delivered simultaneously orsequentially so that the cellularly translated component-specificimmunostimulating agents and externally added elements work in concertto specifically target the immune response.

An especially preferred embodiment provides methods for activation ofthe immune response using vector compositions comprising agentscomprised of a specific antigen in combination with a component-specificimmunostimulating agent. Such methods are effective and can be used inin vitro or in vivo. As used herein, component-specificimmunostimulating agent means an agent that is specific for a componentof the immune system, such as a B or T cell, and that is capable ofaffecting that component, so that the component has an activity in theimmune response. The component-specific immunostimulating agent may becapable of affecting several different components of the immune system,and this capability may be employed in the methods and compositions ofthe present invention. The agent may be naturally occurring or can begenerated or modified through molecular biological techniques or proteinreceptor manipulations.

The activation of the component in the immune response may result in astimulation or suppression of other components of the immune response,leading to an overall stimulation or suppression of the immune response.For ease of expression, stimulation of immune components is describedherein, but it is understood that all responses of immune components arecontemplated by the term stimulation, including but not limited tostimulation, suppression, rejection and feedback activities.

The immune component that is affected may have multiple activities,leading to both suppression and stimulation or initiation or suppressionof feedback mechanisms. The present invention is not to be limited bythe examples of immune responses detailed herein, but contemplatescomponent-specific effects in all aspects of the immune system.

The activation of each of the components of the immune system may besimultaneous, sequential, or any combination thereof. In one embodimentof a method of the present invention, multiple component-specificimmunostimulating agents are administered simultaneously. In thismethod, the immune system is simultaneously stimulated with multipleseparate preparations, each containing a vector composition comprising acomponent-specific immunostimulating agent. Preferably, the vectorcomposition comprises the component-specific immunostimulating agentassociated with the colloidal metal. More preferably, the compositioncomprises the component-specific immunostimulating agent associated withthe colloidal metal of one sized particle or of different sizedparticles and an antigen. Most preferably, the composition comprises thecomponent-specific immunostimulating agent associated with the colloidalmetal of one sized particle or of differently sized particles, anantigen and PEG or PEG derivatives, preferably thiol-PEG (PEG(SH)_(n)),or derivatized poly-l-lysine, preferably poly-l-lysine thiol(PLL(SH)_(n)).

Component-specific immunostimulating agents provide a specificstimulatory, up regulation, effect on individual immune components. Forexample, Interleukin-1β (IL-1β) specifically stimulates macrophages,while TNF-α (Tumor Necrosis Factor alpha) and Flt-3 ligand specificallystimulate dendritic cells. Heat killed Mycobacterium butyricum andInterleukin-6 (IL-6) are specific stimulators of B cells, andInterleukin-2 (IL-2) is a specific stimulator of T cells. Vectorcompositions comprising such component-specific immunostimulating agentsprovide for specific activation of macrophages, dendritic cells, B cellsand

T cells, respectively. For example, macrophages are activated when avector composition comprising the component-specific immunostimulatingagent IL-1β is administered. A preferred composition is IL-1β inassociation with colloidal metal, and a most preferred composition isIL-1β in association with colloidal metal and an antigen to provide aspecific macrophage response to that antigen.

Vector compositions can further comprise targeting molecules,integrating molecules, PEGs or derivatized PEGs.

Many elements of the immune response may be necessary for an effectiveimmune response to an antigen. An embodiment of a method of simultaneousstimulation is to administer four separate preparations of compositionsof component-specific immunostimulating agents comprising 1) IL-1β formacrophages, 2) TNF-α and Flt-3 ligand for dendritic cells, 3) IL-6 forB cells, and 4) IL-2 for T cells. Each component-specificimmunostimulating agent vector composition may be administered by anyroute known to those skilled in the art, and may use the same route ordifferent routes, depending on the immune response desired.

In another embodiment of the methods and compositions of the presentinvention, the individual immune components are activated sequentially.For example, this sequential activation can be divided into two phases,a primer phase and an immunization phase. The primer phase comprisesstimulating APCs, preferably macrophages and dendritic cells, while theimmunization phase comprises stimulating lymphocytes, preferably B cellsand T cells. Within each of the two phases, activation of the individualimmune components may be simultaneous or sequential. For sequentialactivation, a preferred method of activation is administration of vectorcompositions that cause activation of macrophages followed by dendriticcells, followed by B cells, followed by T cells. A most preferred methodis a combined sequential activation comprising the administration ofvector compositions which cause simultaneous activation of themacrophages and dendritic cells, followed by the simultaneous activationof B cells and T cells. This is an example of methods and compositionsof multiple component-specific immunostimulating agents to initiateseveral pathways of the immune system.

One method of binding an agent to metal sols comprises the followingsteps, though for clarity purposes only, the methods disclosed refer tobinding a single agent, TNF, to a metal sol, colloidal gold. Anapparatus was used that allows interaction between the particles in thecolloidal gold sol and TNF in a protein solution. A schematicrepresentation of the apparatus is shown in FIG. 11. This apparatusmaximizes the interaction of unbound colloidal gold particles with theprotein to be bound, TNF, by reducing the mixing chamber to a smallvolume. This apparatus enables the interaction of large volumes of goldsols with large volumes of TNF to occur in the small volume of a Tconnector. In contrast, adding a small volume of protein to a largevolume of colloidal gold particles is not a preferred method to ensureuniform protein binding to the gold particles. Nor is the oppositemethod of adding small volumes of colloidal gold to a large volume ofprotein. Physically, the colloidal gold particles and the protein, TNFare forced into a T-connector by a single peristaltic pump that drawsthe colloidal gold particles and the TNF protein from two largereservoirs. To further ensure proper mixing, an in-line mixer is placedimmediately down stream of the T-connector. The mixer vigorously mixesthe colloidal gold particles with TNF, both of which are flowing throughthe connector at a preferable flow rate of approximately 1 L/min.

Prior to mixing with the agent, the pH of the gold sol is adjusted to pH8-9 using 1 N NaOH. A preferred method for adjusting the pH of the goldsol uses 100 mM TRIS to adjust the pH of the colloidal gold sol to pH 6.Highly purified, lyophilized recombinant human TNF is reconstituted. Apreferred method for diluting TNF is in water that has been adjusted topH 6 with 100 mM TRIS. Before adding either the sol or TNF to theirrespective reservoirs, the tubing connecting the containers to theT-connector is clamped shut. Equal volumes of colloidal gold sol and TNFsolution are added to the appropriate reservoirs. Preferredconcentrations of the active agent in solution range from approximately0.01 to 15 μg/ml, and can be altered depending on the desired ratio ofthe agent to metal sol particles. Preferred concentrations of TNF in thesolution range from 0.5 to 4 μg/ml and the most preferred concentrationof TNF for the TNF-colloidal gold composition is 0.5 μg/ml.

Once the solutions are properly loaded into their respective reservoirs,the peristaltic pump is turned on, drawing the agent solution and thecolloidal gold solution into the T-connector, through the in-line mixer,through the peristaltic pump and into a collection flask. The mixedsolution is stirred in the collection flask for an additional hour ofincubation.

In compositions comprising PEG, whether derivatized or not, the methodsfor making such compositions comprise the following steps, though forclarity purposes only, the methods disclosed refer to adding PEG thiolto a metal sol composition. Any PEG, derivatized PEG composition or anysized PEG compositions or compositions comprising several differentPEGs, can be made using the following steps. Following the 15-minuteincubation, a thiol derivatized polyethylene glycol (PEG) solution isadded to the colloidal gold/TNF sol. The present invention contemplatesuse of any sized PEG with any derivative group, though preferredderivatized PEGs include mPEG-OPSS/2,000, mPEG-OPSS/5,000,mPEG-OPSS/10,000, mPEG-OPSS/12,000, mPEG-OPSS/20,000,mPEG-OP(SS)₂/2,000, mPEG-OP(SS)₂/3,400; mPEG-OP(SS)₂/8,000,mPEG-OP(SS)₂/10,000, thiol-PEG-thiol/2,000, mPEG-thiol 5,000, and mPEGthiol 10,000, mPEG thiol 12,000, mPEG thiol 20,000 (Sun-BIO Inc.). Apreferred PEG is mPEG-thiol 5000 at a concentration of 150 μg/ml inwater, pH 5-8. Thus, a 10% v/v of the PEG solution is added to thecolloidal gold-TNF solution. The gold/TNF/PEG solution is incubated foran additional 15 minutes.

In a preferred method, the TNF and PEG-THIOL moiety simultaneously bindsto the colloidal gold nanoparticle. In this method the pH of thecolloidal gold nanoparticles is adjusted to 6.0 using 100 mM TRIS Base.Similarly the pH of water is adjusted to 6.0 using the 100 mM TRISsolution. Into the latter solution TNF and PEG-THIOL (20,000) arediluted to a final concentration of 5 and 15 ug/ml, respectively. Boththe colloidal gold nanoparticles and TNF/PEG-THIOL solutions are loadedinto their respective reservoirs and bound through the T-connector andin-line mixer using a peristaltic pump to draw each solution through theT-connector. After binding for 15 minutes Human Serum Albumin (200 μg/mlin H₂O) is added to the colloidal gold/TNF/PEG-THIOL solution andincubated for an additional 15 minutes.

The colloidal gold/TNF/PEG solution is subsequently ultrafilteredthrough a 50-100K MWCO diafiltration cartridge. The 50-100K retentateand permeate are measured for TNF concentration by ELISA to determinethe amount of TNF bound to the gold particles.

After diafiltration, cryoprotectants, such as a compositions ofmannitol, 20 mg/ml; and/or human serum albumin, 5 mg/ml, are added andthe samples frozen at −80° C. The samples are lyophilized to dryness andsealed under a vacuum, subsequently reconstituted and analyzed for theamount of free and colloidal gold bound TNF present in the reconstitutedsamples.

The compositions of the present invention can be administered to invitro and in vivo systems. In vivo administration may include directapplication to the target cells or such routes of administration,including but not limited to formulations suitable for oral, rectal,transdermal, ophthalmic, (including intravitreal or intracameral) nasal,topical (including buccal and sublingual), vaginal or parenteral(including subcutaneous, intramuscular, intravenous, intradermal,intratracheal, and epidural) administration. A preferred methodcomprising administering, via oral or injection routes, an effectiveamount of a composition comprising vectors of the present invention.

The formulations may conveniently be presented in unit dosage form andmay be prepared by conventional pharmaceutical techniques.Pharmaceutical formulation compositions are made by bringing intoassociation the metal sol vectors and the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the compositions with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLES Example 1 Manufacture of Colloidal Gold

Colloidal gold sols are manufactured using the reaction described byFrens and Horisberger (Frens, G. Nature Phys. Sci. 1972, 241, 20-22, andHorsiberger, M. Biol. Cellulaire. 1979. 36: 253-258). In this reactionionic gold, in the form of HAuCl₄, is reduced to nanoparticles of Au⁰ bythe addition of sodium citrate. Typically, 2.5 ml of a 4% chloroauricacid (in water) solution is added to 1 L of deionized water. Thesolution is vigorously stirred and heated to a rolling boil. Thereduction reaction is initiated by the addition of a 1% sodium citratesolution. The size of the particle is controlled by the amount ofcitrate added to the reaction. For example, 17, 32, and 64 nm particlesare formed by the addition of 40, 15, and 7.5 ml of the citratesolution, respectively. After the addition of citrate, the solution isallowed to boil and mix for an additional 45 minutes. Upon cooling, thesol is filtered through a 0.22 μm sterilization filter and stored atroom temperature until used.

The production of colloidal gold sols has been scaled-up from 1.0 L to10 L. UV-VIS wavelength scans, dynamic light scattering, anddifferential centrifugation techniques are used to check these particlesfor average particle size and homogeneity. Manufactured particles have amean particle size that routinely measures within 10% of their predictedsize and exhibit a poly-dispersity measure of 1.03-1.12 or less.

Example 2 Increasing the Number of Immune Competent B Cells

To increase the number of immune competent B cells for immunization, MHCclass II restricted-surface IgM⁺/sIgD⁺ human B cells are isolated fromunits of whole blood or buffy coats. Magnetic beads coated withanti-IgM, anti-IgD and anti-CD19 antibodies separate the B cellpopulations. Treating sIgM⁺/sIgD⁻ immature B cells with the cytokineinterleukin-7 is used to recruit additional B cells (Sudo, T., Ito, M.,Ogawa, Y., Iizuka, M., Kodoma, H., Kunisasa, T., Hayashi, S. C., Ogawa,M., Sakai, K., Nishikawa, S., Nishkawa, S. C. J. Exp. Med. 1989. 170:333-338). This treatment has been shown to mature these B cells assignaled by the phenotype conversion of sIgM⁺/sIgD⁻ B cells tosIgM⁺/sIgD⁺ B cells. These isolated cells are purified to nearhomogeneity using FACS separation.

Conjugating TNF to carriers such as KLH or thyroglobulin (see discussionbelow) enhances the antigenicity of human TNF. TNF:KLH antigen is boundto the surface of colloidal gold particles which contain a B celltargeting/activating agent such as interleukin-6 (IL-6). IL-6 is acytokine known to stimulate the synthesis of antibodies from immunized Bcells. Having both moieties on the same particle of gold, ensures that Bcells receive the KLH:TNF antigen signal as well as the IL-6 signal toactivate the antibody response.

Example 3 Differentiation of the Primary Antibody Response

Critical to the production of a therapeutic antibody is the process ofclass switching. The primary antibody response from immunized human Bcells results in the secretion of IgM antibodies. A second class oflymphoid cells, known as antigen presenting cells (APCs), alsointernalizes the antigen. Once internalized these cells process theprotein antigen into fragments, which are then expressed on the cell'ssurface bound to one of two major histocompatibility complexes (MHCs).

Depending on the microenvironment, APCs expressing antigen bound toclass II MHC molecules activate one of two subsets of CD4⁺ T cells.These cells, also known as helper T cells, perform the necessaryaccessory functions to facilitate the cellular or the humoral (antibody)immune response. T_(H)1 CD4⁺ cells facilitate the cellular immuneresponse, while the T_(H)2 subset of CD4⁺ cells interact with IgMsecreting B cells to initiate the process of class switching.

The activation of CD4⁺ T_(H)2 T cells by the APC occurs with theformation of a bicellular cleft known as the immune synapse. Theformation of the immune synapse involves interaction and rearrangementof signaling and structural ligands on the APC with their respectivereceptors on the T cell to form a three-dimensional (3-D) bridge thatallows contact and signaling between these two cells (FIG. 1). Antigensignaling between the APC and the T cell occurs through the binding ofthe MHC/antigen complex with the T cell receptor complex, while thestructural integrity of the immune synapse is maintained by theinteraction of ICAM, LFA-3, and CD72 on the APC with LFA-1, CD2, and CD5receptors on T cells, respectively. The successful formation of theimmune synapse causes the CD4⁺ T cell to express a B cell stimulatorymolecule known as CD40 ligand.

The formation of the immune synapse may signal the T cell to becomeactive or inactive (anergic). Which response is initiated is dependenton the strength of the co-stimulatory signals provided by the B7molecule on the APC to the T cell. The B7 molecule may interact witheither B7 receptor molecule on the T cell, CD28 or CTLA₄. These B7receptors differ with respect to their density on the surface of the Tcell as well as their affinity for the B7 molecule. CD28 has a loweraffinity for B7 than CTLA₄, but is present at a much higher density onthe surface of the T-cell. The binding of B7 to the CD28 receptor sendsan activation signal to the T cell while the binding of B7 by CTLA₄induces T cell anergy (Kuby, J., Immunology Third Edition 1997. edsAllen D., pp-213-218). Thus presenting excess B7 in the immune synapsewill ensure that the T cells will be activated.

In this process immunized human B cells undergo rearrangement of theimmunoglobulin genes to produce highly specific high affinity IgGantibodies.

This vector is initially assembled from MHC, B7, and ICAM proteins ontothe surface of colloidal gold particles. The presentation of the immunesynapse is in the 3-D orientation to allow this vector to successfullytrigger CD4⁺T-cells to express CD40 ligand in an MHC-restricted fashion.

This process is also optionally initiated by using the sTc thatexpresses CD40 Ligand in combination with various cytokines and thesynthetic germinal center whose multiple molecules signal the affinitymaturation critical to a therapeutic mAb.

Example 4 Creation of sAPC/sTc/sGC with Spacer Arms

This sAPC is built on streptavidin colloidal gold particles that areused to bind biotinylated forms of the MHC, B7, and ICAM proteins. Thissingle particle sAPC has a greater degree of flexibility, since theconstituent proteins are bound to the colloidal gold particle indirectlythrough biotinylated spacer arms that form a biotin-avidin bridge.Similarly, the sTc and sGCs may be generated using a similar strategyfor tethering their respective components to the colloidal metal.

Example 5 Self-Assembling APCs/sTcs/sGCs

Self-assembling synthetic APCs are developed. Binding each APC proteinto a different colloidal gold particle creates a complex matrix ofimmune synapse proteins. To direct the assembly of this sAPC, sitedirected molecular scaffolds are made to better orient the variousparticles in 3-D. Shown in FIG. 5 is a representation of thisself-assembling sAPC. The formulation of each particle subunit allowsfor a single particle to bind multiple reagents. For illustrationpurposes the MHC class II molecule is bound to a 32 nm colloidal goldparticle that is also bound with streptavidin. The remaining twosubunits of the sAPC, the B7 and ICAM, are bound to 17 nm particles.Like the MHC particle the ICAM subunit contains streptavidin-dockingsites. To assemble this particle biotinylated human serum albumin isused to join the ICAM and MHC particles together. To complete theassembly of the vector, dithiolated polyethylene glycol is used to linkthe MHC and B7 particles together.

In this model, the formation of the immune synapse occurs through T-cellreceptor/membrane rearrangements. This vector may also be bound to asolid support stage such as an EIA plate. These scaffolds allow bothcolloidal gold-targeted antigens and sAPCs present in the same matrix.As a result, upon immunization of the naïve B-cell the sAPC may activatethe CD4 cell to express CD40 ligand and as a result induce classswitching.

By changing the binding partner to CD40L/cytokine or BLYS/CD30L theself-assembling synthetic T cells or synthetic germinal centers aregenerated.

Example 6 Binding of Proteins to Colloidal Gold Particles

The binding of proteins to colloidal gold particles is influenced by thepH of the colloidal gold sol and protein solutions. At an optimal pH,proteins bind to the surface of colloidal gold particles and preventtheir precipitation by salts. Salt-induced precipitation of thecolloidal gold is easily documented by the changes in the color of thesol from red to black. The pH binding optimum is determined for eachprotein described, including the MHC, B7, ICAM, IL-6 and the KLH:TNFantigen. As an example, the procedure described below outlines themethod for binding the MHC molecule to the colloidal gold particles. Asimilar procedure will be used to determine the binding conditions foreach of the other proteins

The pH binding optimum for MHC binding to colloidal gold is determinedby adjusting the pH of 1 ml aliquots of colloidal gold from 4-11 with 1NNaOH. 100 μl aliquots from each of the gold solutions are placed intomicro-centrifuge tubes and incubated for 30 minutes with 1 ng of the MHCprotein. 100 μl of a 10% NaCl solution is then added to each tube. ThepH binding optimum is defined as the pH that allows the MHC protein tobind to the colloidal gold particles, while preventing salt-inducedprecipitation.

In addition to determining the pH binding optimum, a saturation bindinganalysis is performed for each protein. For this test the pH of thecolloidal gold particles will be adjusted to the pH binding optimum asdescribed above. Subsequently increasing amounts (0.025-5 ng of protein)of the MHC protein is added to the 100 μl aliquots of colloidal gold.After binding for 30 minutes, the various aliquots are centrifuged at10,000 rpms to separate free from colloidal gold bound protein. Thesupernatant and colloidal gold pellets are analyzed for the relativeamount of MHC protein present in each fraction.

Example 7 Quantification of the Mass of the MHC Protein Bound

To quantify the mass of the MHC protein bound per particle of gold,quantitative EIAs are developed for the measurement of the MHC and B7proteins. EIAs for ICAM are already commercially available. The MHC andB7 proteins are quantitatively measured by developing a competitivebinding EIA for each protein. Commercially available antibodies to B7and MHC proteins (both antibodies are available from ResearchDiagnostics, Inc.) are coated onto EIA plates using acarbonate/bicarbonate buffer at pH 9.6. MHC and B7 reference standardsare generated to provide a dose range of 1.56 ng/ml to 500 ng/ml. Thesestandards are added to the EIA plate containing specific antibodies foreither the MHC or B7 protein. The colloidal gold bound samples are addedto other designated wells in the EIA plate.

The concentration of the various proteins is determined by establishinga competitive binding reaction between the protein present in the sampleor standard and a biotinylated form of the molecule for antibody sites.The biotinylated ligand is detected with streptavidin alkalinephosphatase. Upon the addition of substrate, an inverse relationship isgenerated between the mass of analyte present in the sample and theamount of color developed.

Example 8 Binding Multiple Proteins to the Same Particle

To increase the efficiency and specificity of the in vitro immunizationmultiple chemically distinct proteins need to be bound onto the surfaceof a single colloidal gold particle. The binding of three differentprotein cytokines (IL-1, IL-6 and TNF) to the same particle of colloidalgold is demonstrated. Each cytokine binds to colloidal gold at aspecific pH.

As demonstrated above, it was determined that IL-1 bound to colloidalgold at a pH between 6 and 8 while TNF and IL-6 bound at a pH of 8 and11, respectively. A solution containing 0.25 ng/ml of the threecytokines in water was mixed with a colloidal gold sol at pH 8. A samplewas removed and the pH of the remaining solution was adjusted to 11.Prior to each pH change additional samples were collected. The twosamples were centrifuged and the resultant pellets of colloidal goldwere re-suspended in PBS.

To demonstrate the presence of all three cytokines on the same particleof gold the various pellets were added to an EIA plate that was coatedwith a monoclonal antibody to TNF. After binding, the plate was washedand designated wells were incubated with either an alkaline phosphataseconjugated rabbit anti IL-1, IL-6, or TNF. After a wash, substrate wasadded to each well to initiate color development. The data presented inFIG. 6 show that due to the overlap in pH binding optimum both IL-1 andTNF were present on the particle at pH 8. However, very little IL-6signal could be detected. Increasing the pH 11 allowed IL-6 to bind tothese particles.

Example 9 Targeting of Chimeric Vectors to Specific Cells

EGF and streptavidin were bound to the same 32 nm particle of colloidalgold. The sample was divided into three aliquots for the binding ofsecondary/targeting molecules. One sample was bound with biotinylatedIL-1, another biotinylated GM-CSF, and the third with biotinylated IL-6.After binding the biotinylated ligands, the samples were centrifuged toremove any free reagents and the colloidal gold pellets were added toFicoll-separated human white blood cells. After 8 days in culture theuptake of the various colloidal gold vectors was documented by digitalphotography.

EGF streptavidin gold was targeted to macrophages (FIG. 7A), dendriticcells (FIG. 7B) and B-Cells (FIG. 7C) by using biotinylated IL-1,biotinylated GM-CSF, or biotinylated IL-6 for targeting. The blackstaining (highlighted by the red arrows) in each of the figuresrepresents the uptake of the various colloidal gold vectors.

As can be seen in FIG. 7, the various colloidal gold/cytokine chimerasdifferentially targeted the various cellular elements of the immunesystem. The black staining (highlighted by the arrows) represents thecolloidal gold particles, which have been internalized and aggregated bythe various immune cells. These data indicate that IL-1 targeted thecolloidal gold EGF to macrophages, while GM-CSF targeted the chimera todendritic cells, and IL-6 targeted the vector to B cells.

Example 10 Immunization of Human Lymphocytes

These vectors can be used to generate a primary immune response fromisolated lymphocytes. White blood cells were collected from whole bloodby density centrifugation. These cells were treated with a thyroglobulinconjugated TNF/IL-6 colloidal gold vector. The cells received pulses ofthe colloidal gold vector every 2 days for a total of eight days. Afterthe final pulse, the cells were cultured for another 5 days. Thesupernatants were collected and tested for the presence of humananti-human TNF (IgM/IgD and IgG combination) antibodies using a directEIA. As can be seen in FIG. 8, the chimeric cAU thyroglobulin TNF hadthe highest immunodensity.

Example 11 Immunization of Human B-Cells and Dendritic Cells for ClassII MHC Expression

Two different approaches to increase the efficiency of in vitro humanlymphocyte immunization are used. First, coupling TNF to immunogeniccarriers, such as Thyroglobulin, Keyhole Limpet Hemocyanin or MurineSerum Albumin enhances TNF's immunogenicity. Carrier:TNF conjugationsare performed using standard EDC/NHS and gluteraldehyde methods. Second,coupling them to particles of colloidal gold, containing cell-specifictargeting agents increases the specificity of these antigens. To targetthe delivery of the antigen to B cells the carrier:antigen complex isbound to particles of colloidal gold particles containing IL-6. Totarget the delivery of the carrier antigen to dendritic cells thecarrier:antigen complex is bound to colloidal gold particles containingGM-CSF.

These vectors are initially used to immunize naïve MHC restricted humanB cells and dendritic cells for the generation of the class II MHCantigen.

These same vectors are used at a later time to induce the primaryantibody response from a new or replicate set of naïve B cells. Theimmunization scheme involves the sequential immunization of B cells anddendritic cells with the various vectors. As a result the B cells anddendritic cells see the carrier once and the TNF antigen three times.

Example 12 Generation of Class II MHC Protein by B Cells

To cause human B-cells to produce class II MHC protein, 10⁶ surfaceIgM⁺/IgD⁺ human B-cells are plated in 24-well plates and cultured in 1.5ml of AIM V media. Twenty four hours after plating, the cells are pulsedwith the THYRO:TNF antigen bound to an IL-6 targeted colloidal goldvector. Two days later the cells are pulsed with the KLH:TNF carriertargeted by the IL-6 vector. After an additional two days in culture thecells are immunized with the third carrier:TNF antigen, MSA:TNF. Thecells are incubated for an additional three to seven days and tested forthe presence of Class II MHC expression by FACS analysis. Alternatively,the cells may be simultaneously pulsed with the colloidal gold antigens.

A similar procedure is used to pulse dendritic cells to express the MHCclass II protein. These cells are immunized with the TNF:carrier antigenbound to GM-CSF targeted colloidal gold vectors. Dendritic cellprecursors are isolated from peripheral blood using magnetic beadscoated with anti-CD34. These cells are expanded in vitro by incubatingthem in AIM V serum free media supplemented with 1000 ng/ml of GM-CSFand 100 ng/ml of IL-4. Upon their maturation, confirmed by FACS analysisfor the detection of CD1a and empty class II MHC molecules, the cellsare differentiated into mature dendritic cell with a 10 ng/ml pulse ofTNF. These mature dendritic cells are immunized with the GM-CSF targetedcolloidal gold TNF antigen. Antigen loaded MHC class II proteincomplexes are detected by FACS or in situ analysis of the biotinylatedantigen peptide detected with a streptavidin conjugated phycoerythrin(Research Diagnostic Inc.) detection system.

Example 13 Method Development for the Isolation of the MHC Class IIAntigen

The method for the isolation of the MHC uses “generic”-non-MHCcompatible blood samples. These MHC molecules are used to define the pHand saturation optima for the protein on colloidal gold particles. Oncedefined, these methods are adapted to purify antigen loaded MHC fromimmunized MHC restricted blood pools.

The isolation of generic and antigen loaded human class II MHC is doneusing the method described by Sette (Sette et al., J. Immunol. 1992.148: 844). Briefly the buffy coats from non-HLA matched human wholeblood are frozen at a minimum density of 10⁸ cells/ml and sonicated todisrupt the cells. These cells are suspended in a buffer of 50 mMTRIS-HCl, pH 8.5 with 2% Renex, 150 mM NaCl, 5 mM EDTA and 2 mM PMSF.Large particulates including the nuclei are removed by centrifugation(10000×g for 20 minutes). The cell lysate is then fractionated on anaffinity column made by binding murine antibodies to the human class IIMHC molecule (Research Diagnostics Inc.) to protein A/G sepharose beads.The lysate is passed through the column at least 5 times to maximize thebinding of the MHC protein to the immobilized antibody. The column iswashed with 10 column volumes of a buffer containing 10 mM TRIS-HCl pH8.0/0.1% Renex followed by an additional wash of 5 column volumes of PBSwith 1% n-ocytlglucoside. The MHC class II protein is eluted from thecolumn using a buffer of 50 mM diethylamine in saline with 1%n-ocytlglucoside at a pH 11.5. Upon elution each fraction is immediatelyneutralized with the addition of 2 M glycine, pH 2.0. The fractionscontaining the MHC II molecules are aliquoted and lyophilized in 25 μgaliquots.

Example 14 Generation of Human B7.1 Molecule

The human co-stimulatory molecule B7.1 is made by recombinant DNAtechnology. The gene is supplied as part of a commercially availabletransient expression vector system (InVivogen Inc.). The construct isprovided with the appropriate restriction sites allowing for theseparation of the active gene from the plasmid construct. The humanB-7.1 gene is isolated from the pORF host plasmid using the restrictionenzyme NcoI and NheI. This double digestion results in the formation oftwo linearized pieces of DNA. One of the gene fragments consists of theB-7.1 gene (893 bp) while the other fragment (3210 bp) constitutes theaccessory genes of the p-ORF plasmid. The gene fragments arefractionated on a 1% agarose gel and visualized by ethidium bromidestaining. The bands are cut from the gel and purified using QuiaQuickgel extraction resin. The purified linearized gene is inserted into abaculovirus expression system (CloneTech Inc.) under the control of thestrong CMV promoter. The baculovirus incorporated genes are transfectedinto the SF9 insect cell line according to the manufacturersspecifications and conditions. 10⁶ B7 transfected NOS cells will beexpanded in bioreactors. The incubation media and cell lysates areprocessed by affinity chromatography using a murine monoclonal antibodyagainst the human B7.1 protein (Research Diagnostics Inc.) previouslyimmobilized to a protein A/G sepharose column.

Example 15 Generation of the Synthetic Antigen Presenting Cell TheSingle Particle sAPC

To mature the primary antibody response the sAPC capable must induce theCD4 T-cell/B-cell interactions that result in antibody class switching.The first sAPC is developed by binding the proteins of the immunesynapse on a single particle of colloidal gold. This vector as well asone built on a streptavidn colloidal gold core are tested for theirability to activate CD4⁺ T-cells.

Once the components of the immune synapse are isolated and purified tohomogeneity they are bound to colloidal gold particles to develop thesingle particle sAPC. Two strategies are used to develop these APCs. Thefirst strategy involves the direct binding of the components of theimmune synapse (i.e., the peptide loaded MHC, B7 and ICAM molecules) toparticles of colloidal gold. While not wishing to be bound, it isbelieved that each ml of gold will bind 250 ng of each protein/ml ofcolloidal gold sol.

The first scaffold was assembled on the surface of an EIA plate. Thematerials include an EIA plate coated with a monoclonal antibody tohuman TNF; a 32 nm TNF/streptavidin colloidal gold chimera; biotinylatedBSA: a 17 nm streptavidin colloidal gold vector; biotinylated humanIL-6, Rabbit anti human IL-6 conjugated to alkaline phosphatase. Thevarious components were assembled into a scaffold as depicted in FIG.9A. The control for this study simply was the 32 nm particle without theTNF docking site upon which the scaffold was built. As presented in FIG.10A a strong signal was generated when all of the molecular bricks ofthe scaffold were present. By merely omitting the TNF docking site thescaffold did not form and as a result no signal was generated.

The direct binding of the immune synapse proteins to a single particleof colloidal gold results in a rigid orientation of the proteins on thesurface of the particle. To increase the flexibility of movement forthese proteins on sAPC an alternative single particle sAPC is developed.This single particle sAPC is developed on a streptavidin colloidal goldplatform that binds biotinylated forms of the MHC, B7, and ICAMproteins. The proteins are bound to the streptavidin gold particlethrough the biotin residue that is linked to the protein through aspacer arm.

The proteins are biotinylated using several biotinylating reagents suchas NHS-Biotin (Pierce Chemical Co.). This reagent places a 1.35 nmspacer arm between the protein and biotin moieties. Alternatively,NHS-LC-LC-Biotin is used to biotinylate the proteins. This agents placea 3.05 nm spacer arm between the protein and biotin residue. Such aspacer arm facilitates movement of the proteins to promote ligandbinding. This added flexibility improves the ability of the proteins toachieve a proper 3-D orientation and to form a functional immune synapsewith the CD4⁺T-cell.

Example 16 Generation of a Self-Assembling sAPC

The multiparticle sAPC will have the flexibility of self orientationduring immune synapse formation. The flexibility is a direct result ofassembling the moieties used to join the particles together. Linkers canbe alkane, protein, and polyethylene glycol (PEG) to allow for thegreatest vector functionality.

The second scaffold (shown in FIG. 9B) was assembled using a four-armPolyethylene glycol (10,000 MW) backbone containing four terminal freethiols. This linker was used to join individual particles of colloidalgold bound with either IL-1 or TNF. After linkage the preparation wascentrifuged and assayed for both proteins using an EIA plate coated onlywith an IL-1 monoclonal antibody. After binding the plate was washed anddetected using enzyme linked IL-1 or TNF polyclonal antibodies.Similarly the vector described in FIG. 9B generated a signal (FIG. 10 B)for both proteins only in the presence of the linker. Without the linkeronly background color was observed.

To further increase the flexibility of the sAPC the component proteinsare assembled on different particles of colloidal gold. These particlesare assembled into a scaffolding system to generate a sAPC capable ofinducing CD4⁺ T-cell activation. The multi-particle sAPC may be used insolution or as is shown in FIGS. 9A and 9B to provide a solid support onan EIA plate.

The MHC, B7 and ICAM proteins are bound to different particles ofcolloidal gold as previously described. The particles are physicallyjoined by a variety of scaffolding molecules. The function of the“joining” molecules is to provide greater flexibility of the individualparticles of colloidal gold in the formation of the immune synapse. Thisflexibility occurs whether the sAPC is provided as an independentparticle or as part of a matrix bound to a solid surface.

The first additive consists of modified di-thiol alkane moieties. Thefunction of alkane di-thiol binds, through the formation of a thiol-goldbond, the individual particles of colloidal gold together. Thesemoieties have been used to build self-assembling gold structures on thesurface of glass slides in the development of biosensors (Mirkin, C. A.,Letsinger, R., Mucic, R. C., and Storhoff, J. J. Nature. 1996. 382607-609). The thiol group allows the binding of the alkane moietydirectly to the surface of the colloidal gold particle. Examples of thecommercially available alkane thiol reagents include: 1,5 pentanedi-thiol, 1,6 hexane di-thiol, and decane di-thiol (Sigma ChemicalCompany).

As an alternative to the alkane di-thiols various sizes of 2, 3 and4-arm poly-ethylene glycol (SunBio, Walnut Creek, Calif.) are also used.Each arm of these polymers has a free thiol group, which is used to bindthe individual particles of colloidal gold through the formation of agold-thiol bound. These reagents provide the added advantage of completesolubility in water.

Binding multiple protein moieties of the immune synapse to either singleor multiple particles of colloidal gold enables the generation of asynthetic antigen-presenting cell (sAPC) capable of driving the cellularevents that cause class switching in immunize human B-cells.

Example 17 Stimulation of CD4⁺T-cells by sAPC to Express CD4⁺ Ligand

Single particles and self-assembling sAPCs are tested for their abilityto induce the expression of CD40 ligand from MHC restricted CD4⁺T-cells. Subsequently, 0.1 to 10 ug of antigen loaded MHC (present onthe sAPC) are added to 10⁶ class II restricted CD4⁺ T cells growing inAIM V media. The stimulation occurs in the presence of IL-4 and IL-10,which drives the production of the T_(H)2 subset of CD4⁺ T cells. After4, 12 and 24 hours of sAPC stimulation the CD4 cells are collected andstained with a FITC labeled mouse anti human CD40 ligand antibody andanalyzed by FACS.

During the activation of the CD4⁺ T cells a new set (i.e., cells notused for the isolation of the MHC) of MHC restricted B cell lymphocytesare immunized as was previously described to undergo the production ofantigen specific IgM antibodies. MHC restricted B cells are immunizedusing the targeted TNF antigens previously described. Upon the detectionof antigen specific IgMs and CD40 ligand production from theirrespective cells the activate CD4⁺ T cells are added to IgM secreting Bcells. Class switching is monitored by the detection human-anti-humanTNF IgGs. IgG positive clones are fused with the K6H6/B5 mouse humanheteromyeloma cell line as described below.

Example 18 Antibody Detection and Immortalization of B Cells

All of the cells from positive wells are combined, centrifuged once,washed with PBS and combined with 2×10⁶ mouse/human heteromyelomaK6H6/B5 cells. The heteromyeloma cell line, K6H6/B5 (available throughthe ATCC), is an ideal fusion partner for these human lymphocytesbecause these cancer cells are non-secretors of antibody and areavailable with no patent restrictions. The human and myeloma cells arefused using standard fusion protocols with PEG. Successfully fused cellsare selected using traditional HAT/HT selection protocols. A directELISA is used to test growing clones for the production of TNF specifichuman IgG antibody. Those clones that show antigen recognition arescaled-up in T-75 flasks, at which point all clones are cryopreservedand their supernatants tested for neutralizing antibody activity asdescribed below.

Example 19 Neutralization of TNF Biologic Activity

The ability of the TNF antibodies to neutralize the biologic activity ofTNF is tested using the well-characterized WEHI 164 bioassay. Briefly,TNF dose-dependently inhibits the in vitro proliferation of these cells.

For this bioassay 5000 WEHI cells are plated in 24-well tissue cultureclusters. TNF (15.6 pg/ml to 500 pg/ml) is added to designated wells inthe plate. To determine the ability of the human monoclonal antibodiesto neutralize the action of TNF an identical standard dose range of TNFstandards is made in the presence of 1 μg of each of the TNF monoclonalantibodies. The cells with the various treatments are cultured for 5days and cell number is determined using a Coulter Counter.

Example 20 Effect of Ionic Strength on the Lyophilization Stability ofColloidal Gold Bound TNF

The colloidal gold binding apparatus, shown in FIG. 11, was used to bindTNF to colloidal gold nanoparticles as previously described. Afterbinding, 30K PEG-Thiol was added to the solution at 50 μg/ml indeionized water, pH 9.

To test the effect of ionic strength on the stability of theTNF-colloidal gold bond various amounts of salt (in the form of 1×normal phosphate buffered saline; PBS) were added to the containerholding the TNF solution. Final concentrations of PBS varied from 0 to0.325% of normal PBS. After binding and diafiltration, cryoprotectants(mannitol, 20 mg/ml; human serum albumin, 5 mg/ml) were added to thesamples. The samples were subsequently aliquoted into 1 ml samples andfrozen at ±80° C. After freezing the samples were lyophilized to drynessand sealed under a vacuum.

Subsequently the samples were reconstituted with 1 ml of deionized waterand diluted ten-fold in a 1% PEG-1450/water solution. The samples werecentrifuged to separate colloidal gold bound TNF from free TNF. Both thecolloidal gold pellets and supernatants were analyzed for TNFconcentrations by EIA. The data from these studies are presented inTable I.

TABLE 1 Release Profile of Lyophilized Colloidal Gold TNF Manufacturedin the Absence of Salt Percent of Total TNF Colloidal Gold Pellet 68Supernatant 32

Table I shows that 32% of the TNF is released from the vector followinglyophilization. In repetitive studies we observed that as much as 50% ofthe protein is released after lyophilization.

Example 21 Effect of Increasing Ionic Strength on the Stability of aLyophilized Colloidal Gold-TNF Drug

The solution of TNF, which was previously diluted in a 3 mM TRISsolution to a final concentration of TNF of 0.5 μg/ml, was modified byadding 0.25× solution (77.25 milli-osmol/kg) of normal phosphatebuffered saline. The solution was bound as was described above. Afterbinding, 30K PEG-Thiol and the cryoprotectants described above wereadded and the samples frozen at −80° C. The samples were lyophilized asdescribed above, subsequently reconstituted and analyzed for the amountof free and colloidal gold bound TNF present in the reconstitutedsamples. The data from this study are presented in FIG. 12.

As can be seen in FIG. 2, increasing the ionic strength significantlyimproves the stability of the vector during lyophilization. The saltingeffect was dose dependent. As shown in Table II as the amount of saltadded to the TNF is decreased more of the protein is released afterlyophilization.

TABLE II Effect of Salt Concentration on TNF Release Following VectorLyophilization Salt Concentration (milli-osmol/kg) 0 4.5 19.3 46.5 77.25Percent Released 40 30 10 6 6

Binding TNF in the absence of salt results in a portion of the TNF beingbound in the ionic double layer rather than directly on the goldparticle (FIG. 13). During lyophilization water solvating the ioniclayer is lost and thus upon reconstitution this vector released theportion of TNF bound in the ion cloud. By increasing the ionic strengththrough the addition of salt the ionic layer shrinks/collapses (FIG. 14)and allows for all of the TNF to bind directly to the particles'surface. After lyophilization, this preparation has all of the TNF boundto the particles' surface.

All patents, publications and abstracts cited above are incorporatedherein by reference in their entirety. It should be understood that theforegoing relates only to preferred embodiments of the present inventionand that numerous modifications or alterations may be made thereinwithout departing from the spirit and the scope of the present inventionas defined in the following claims.

1. A composition, comprising a synthetic antigen presenting cell (APC),wherein the synthetic APC comprises a major histocompatibility complex(MHC) protein bound to a first colloidal metal particle, a structuralprotein bound to a second colloidal metal particle, and a co-stimulatoryprotein B7 bound to a third colloidal metal particle, wherein the first,second and third colloidal metal particles are bound to each other byscaffolding molecules.
 2. The composition of claim 1, wherein the MHCprotein, the structural protein and the co-stimulatory protein B7 arebound indirectly by a binding pair.
 3. The composition of claim 2,wherein the binding pair comprises streptavidin-biotin.
 4. Thecomposition of claim 1, wherein the structural protein is selected fromthe group consisting of: intracellular adhesion molecule (ICAM), LFA-3,and CD72.
 5. The composition of claim 1, wherein the colloidal metalcomprises colloidal gold, colloidal silver, colloidal iron, colloidalaluminum, or colloidal platinum.
 6. The composition of claim 1, furthercomprising a pharmaceutically-acceptable component comprisingexcipients, buffers or carriers.
 7. The composition of claim 1, furthercomprising an adjuvant, wherein the adjuvant comprises liposomes,emulsions, microspheres, biodegradable polymers and polystyrene, alum,heat killed M. butyricum and M. tuberculosis, Pertussis toxin andTetanus toxin, or LPS and Staphylococcal enterotoxin B.
 8. Thecomposition of claim 1, wherein the MHC protein is an antigen-loaded MHCprotein.
 9. The composition of claim 1 wherein the colloidal metalparticles are the same size.
 10. The composition of claim 1, wherein thecolloidal metal particles are different sizes.
 11. The composition ofclaim 10, wherein the first colloidal metal particle is 32 nanometers(nm), and the second and third colloidal metal particles are 17 nm. 12.The composition of claim 1, wherein the first, second and thirdcolloidal metal particles are coated with streptavidin and wherein thescaffolding molecule is a biotinylated protein.
 13. The composition ofclaim 12, wherein the biotinylated protein is human serum albumin (HSA).14. The composition of claim 1, wherein the scaffolding molecule is adi-thiol alkane.
 15. The composition of claim 1, wherein the scaffoldingmolecule is a 2 or 4-arm poly-ethylene glycol (PEG).